Poly ADP-ribose polymerase gene and its uses

ABSTRACT

Compositions and methods for influencing the metabolic state of plant cells are provided. The compositions comprise poly ADP-ribose polymerase genes and portions thereof, particularly the maize poly ADP-ribose polymerase gene as well as antisense nucleotide sequences for poly ADP-ribose polymerase genes. The nucleotide sequences find use in transforming plant cells to alter the metabolic state of the transformed plants and plant cells.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional Application No.60/072,785, filed Jan. 27, 1998.

FIELD OF THE INVENTION

The invention is drawn to the genetic manipulation of plants.

BACKGROUND OF THE INVENTION

The physiological and metabolic state of plant cells directly influencesthe plant response to external stimuli. The plant response to diseaseincludes a host of cellular processes to enable plants to defendthemselves from pathogenic agents. These processes apparently form anintegrated set of resistance mechanisms that is activated by initialinfection and then limits further spread of the invading pathogenicmicroorganism.

The transformation of plants is a complex process. The process involvescontacting cells with a DNA to be integrated into the plant cell genome.Generally, genetic transformation of eukaryotic cells is a random event.That is, the foreign DNA is integrated into the genome at randompositions. Often several copies, or parts of copies, of the transformingDNA are integrated in a single position, and/or at different positions,resulting in a transformed cell containing multiple copies of theforeign DNA.

Because the metabolic state of the plant cell is instrumental in variousprocesses, it would be beneficial to be able to influence the state ofthe cells. Accordingly, there is a need for genes and methods foraltering the metabolic state of plant cells.

SUMMARY OF THE INVENTION

Compositions and methods for influencing the metabolic state of plantcells are provided. The compositions comprise poly ADP-ribose polymerasegenes and fragments thereof, particularly the maize poly ADP-ribosepolymerase gene. The genes or antisense constructions of the genes canbe used to transform plant cells and alter the metabolic state of thetransformed cell.

In this manner, transformed plants can be obtained having alteredmetabolic states. The invention has implications in enhancing diseaseresistance in plants and for methods of genetic transformation ofplants.

DETAILED DESCRIPTION OF THE INVENTION

Poly ADP-Ribose Polymerase genes and methods for their use are provided.In particular, the amino acid and nucleotide sequences for the maizepoly ADP-ribose polymerase (PARP) are provided as SEQ ID NOs. 2 and 1,respectively. Also of interest are portions of the sequences of theinvention. The nucleotide and amino acid sequences of the C-terminaldomain of the maize poly ADP-ribose polymerase is provided in SEQ IDNOs. 3 and 4, respectively. The nucleotide sequence of the Zinc fingersis provided in SEQ ID NO. 5.

PARP is generally described as a nuclear enzyme found in mosteukaryotes. Structure-function studies have shown that animal PARPs maybe divided into at least three subdomains. The N-terminal part containstwo zinc fingers and has a high affinity for nicked V-shaped DNA.Interaction of PARP with nicked DNA strongly enhances the activity ofthe catalytic domain, which is very well conserved among PARPs andlocated in the carboxyl-terminus of the protein. (Ueda et al. (1985)Ann. Rev. Biochem. 54:73-100; Sdhah et al. (1995) Anal. Biochem.227:1-13).

PARP catalyzes both the transfer of ADP-ribose from NAD⁺, mainly to thecarboxyl group of a glutamic acid residue on target proteins, andsubsequent ADP-ribose polymerization. (Ueda et al. (1985) Ann. Rev.Biochem. 54:73-100; Sdhah et al. (1995) Anal. Biochem. 227:1-13)

PARP is generally required in most cases where DNA is cleaved andrejoined, such as in DNA repair, DNA recombination, gene rearrangementsand transposition. PARP has been shown to modify PARP itself, histones,high mobility group chromosomal proteins, topoisomerase, endonucleasesand DNA polymerases. (Ueda et al. (1985) Ann. Rev. Biochem. 54:73-100;Sdhah et al. (1995) Anal. Biochem. 227:1-13).

Initially, the enzyme synthesizes an ester linkage preferentiallybetween the glutamyl(-) or sometimes the C-terminal(-)carboxyl group onthe acceptor protein and the 1′-OH of the ribosyly group of ADP-ribose.Subsequently, up to 45-50 ADP units are added via a 2′-1′phosphodiesterbond. Branching of the poly (ADP)-ribosyl chains via the2′-1′phosphodiester linkages is also observed. See, for example, Ueda etal. (1985) Ann. Rev. Biochem. 54:73-100; and Shah et al. (1995) Anal.Biochem. 227:1-13.

Compositions of the invention include isolated nucleic acid moleculesencoding the PARP proteins of the invention, as well as fragments andvariants thereof. The term “isolated” refers to material, such as anucleic acid or a protein, which is: (1) substantially or essentiallyfree from components that normally accompany or interact with it asfound in its naturally occurring environment. Thus, for a nucleic acid,the sequence is lacking a flanking sequence either 3′ or 5′ or both. Theisolated material optionally comprises material not found with thematerial in its natural environment; or (2) if the material is in itsnatural environment, the material has been synthetically (non-naturally)altered by deliberate human intervention to a composition and/or placedat a locus in the cell (e.g., genome or subcellular organelle) notnative to a material found in that environment. The alteration to yieldthe synthetic material can be performed on the material within orremoved from its natural state. For example, a naturally occurringnucleic acid becomes an isolated nucleic acid if it is altered, or if itis transcribed from DNA which has been altered, by non-natural,synthetic (i.e., “man-made”) methods performed within the cell fromwhich it originates. See, e.g., Compounds and Methods for Site DirectedMutagenesis in Eukaryotic Cells, Kmiec, U.S. Pat. No. 5,565,350; In VivoHomologous Sequence Targeting in Eukaryotic Cells; Zarling et al.,PCT/US93/03868. Likewise, a naturally occurring nucleic acid (e.g., apromoter) becomes isolated if it is introduced by non-naturallyoccurring means to a locus of the genome not native to that nucleicacid. Nucleic acids which are “isolated” as defined herein, are alsoreferred to as “heterologous” nucleic acids.

As used herein, “localized within the chromosomal region defined by andincluding” with respect to particular markers includes reference to acontiguous length of a chromosome delimited by and including the statedmarkers.

As used herein, “marker” includes reference to a locus on a chromosomethat serves to identify a unique position on the chromosome. A“polymorphic marker” includes reference to a marker which appears inmultiple forms (alleles) such that different forms of the marker, whenthey are present in a homologous pair, allow transmission of each of thechromosomes in that pair to be followed. A genotype may be defined byuse of one or a plurality of markers.

As used herein “operably linked” includes reference to a functionallinkage between a promoter and a second sequence, wherein the promotersequence initiates and mediates transcription of the DNA sequencecorresponding to the second sequence. Generally, operably linked meansthat the nucleic acid sequences being linked are contiguous and, wherenecessary to join two protein coding regions, contiguous and in the samereading frame.

The nucleotide sequences of the invention can be used to isolate otherhomologous sequences in other plant species. Methods are readilyavailable in the art for the hybridization of nucleic acid sequences.Coding sequences from other plants may be isolated according to wellknown techniques based on their sequence homology to the codingsequences set forth herein. In these techniques all or part of the maizecoding sequence is used as a probe which selectively hybridizes to otherPARP coding sequences present in a population of cloned genomic DNAfragments or cDNA fragments (i.e. genomic or cDNA libraries) from achosen organism. For example, the entire maize PARP sequence or portionsthereof may be used as probes capable of specifically hybridizing tocorresponding coding sequences and messenger RNAs. To achieve specifichybridization under a variety of conditions, such probes includesequences that are unique and are preferably at least about 10nucleotides in length, and most preferably at least about 20 nucleotidesin length. Such probes may be used to amplify the PARP coding sequencesof interest from a chosen organism by the well-known process ofpolymerase chain reaction (PCR). This technique may be used to isolateadditional coding sequences from a desired organism or as a diagnosticassay to determine the presence of coding sequences in an organism.

Such techniques include hybridization screening of plated DNA libraries(either plaques or colonies; see, e.g., Sambrook et al., MolecularCloning, eds., Cold Spring Harbor Laboratory Press (1989)) andamplification by PCR using oligonucleotide primers corresponding tosequence domains conserved among the amino acid sequences (see, e.g.Innis et al., PCR Protocols, a Guide to Methods and Applications, eds.,Academic Press (1990)). For example, hybridization of such sequences maybe carried out under conditions of reduced stringency, medium stringencyor even stringent conditions (e.g., conditions represented by a washstringency of 35-40% Formamide with 5×Denhardt's solution, 0.5% SDS and1×SSPE at 37 C.; conditions represented by a wash stringency of 40-45%Formamide with 5×Denhardt's solution, 0.5% SDS, and 1×SSPE at 42 C.; andconditions represented by a wash stringency of 50% Formamide with5×Denhardt's solution, 0.5% SDS and 1×SSPE at 42 C., respectively), toDNA encoding the PARP genes disclosed herein in a standard hybridizationassay. See J. Sambrook et al., Molecular Cloning, A Laboratory Manual 2ded. (1989) Cold Spring Harbor Laboratory. In general, sequences whichcode for the defense activators and other activator proteins of theinvention and hybridize to the sequences disclosed herein will be atleast 50% homologous, 70% homologous, and even 85% homologous or morewith the disclosed sequence. That is, the sequence similarity ofsequences may range, sharing at least about 50%, about 70%, and evenabout 85% sequence similarity.

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or polynucleotides: (a) “referencesequence”, (b) “comparison window”, (c) “sequence identity”, (d)“percentage of sequence identity”, and (e) “substantial identity”.

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence.

(b) As used herein, “comparison window” means includes reference to acontiguous and specified segment of a polynucleotide sequence, whereinthe polynucleotide sequence in the comparison window may compriseadditions or deletions (i.e., gaps) compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. Generally, the comparison window is at least 20contiguous nucleotides in length, and optionally can be 30, 40, 50, 100,or longer. Those of skill in the art understand that to avoid a highsimilarity to a reference sequence due to inclusion of gaps in thepolynucleotide sequence a gap penalty is typically introduced and issubtracted from the number of matches.

Methods of alignment of sequences for comparison are well-known in theart. Optimal alignment of sequences for comparison may be conducted bythe local homology algorithm of Smith et al. (1981) Adv. Appl. Math.2:482; by the homology alignment algorithm of Needleman et al. (1970) J.Mol. Biol. 48:443; by the search for similarity method of Pearson et al.(1988) Proc. Natl. Acad. Sci. 85:2444; by computerized implementationsof these algorithms, including, but not limited to: CLUSTAL in thePC/Gene program by Intelligenetics, Mountain View, Calif., GAP, BESTFIT,BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis., USA; theCLUSTAL program is well described by Higgins et al. (1988) Gene73:237-244; Higgins et al. (1989) CABIOS 5:151-153; Corpet et al. (1988)Nucleic Acids Research 16:10881-90; Huang et al. (1992) ComputerApplications in the Biosciences 8:155-65, and Person et al. (1994)Methods of Molecular Biology 24:307-331; preferred computer alignmentmethods also include the BLASTP, BLASTN, and BLASTX algorithms. Altschulet al. (1990) J. Mol. Biol. 215:403-410. Alignment is also oftenperformed by inspection and manual alignment.

(c) As used herein, “sequence identity” or “identity” in the context oftwo nucleic acid or polypeptide sequences includes reference to theresidues in the two sequences which are the same when aligned formaximum correspondence over a specified comparison window. Whenpercentage of sequence identity is used in reference to proteins it isrecognized that residue positions which are not identical often differby conservative amino acid substitutions, where amino acid residues aresubstituted for other amino acid residues with similar chemicalproperties (e.g. charge or hydrophobicity) and therefore do not changethe functional properties of the molecule. When sequences differ inconservative substitutions, the percent sequence identity may beadjusted upwards to correct for the conservative nature of thesubstitution. Sequences which differ by such conservative substitutionsare said to have “sequence similarity” or “similarity”. Means for makingthis adjustment are well-known to those of skill in the art. Typicallythis involves scoring a conservative substitution as a partial ratherthan a full mismatch, thereby increasing the percentage sequenceidentity. Thus, for example, where an identical amino acid is given ascore of 1 and a non-conservative substitution is given a score of zero,a conservative substitution is given a score between zero and 1. Thescoring of conservative substitutions is calculated, e.g., asimplemented in the program PC/GENE (Intelligenetics, Mountain View,Calif., USA).

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity.

(e) (i) The term “substantial identity” of polynucleotide sequencesmeans that a polynucleotide comprises a sequence that has at least 70%sequence identity, preferably at least 80%, more preferably at least 90%and most preferably at least 95%, compared to a reference sequence usingone of the alignment programs described using standard parameters. Oneof skill will recognize that these values can be appropriately adjustedto determine corresponding identity of proteins encoded by twonucleotide sequences by taking into account codon degeneracy, amino acidsequences for these purposes normally means sequence identity of atleast 60%, more preferably at least 70%, 80%, 90%, and most preferablyat least 95%. Polypeptides which are “substantially similar” sharesequences as noted above except that residue positions which are notidentical may differ by conservative amino acid changes.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other under stringent conditions.Generally, stringent conditions are selected to be about 5 C. to about20 C. lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. Typically,stringent wash conditions are those in which the salt concentration isabout 0.02 molar at pH 7 and the temperature is at least about 50, 55,or 60 C. However, nucleic acids which do not hybridize to each otherunder stringent conditions are still substantially identical if thepolypeptides which they encode are substantially identical. This mayoccur, e.g., when a copy of a nucleic acid is created using the maximumcodon degeneracy permitted by the genetic code. One indication that twonucleic acid sequences are substantially identical is that thepolypeptide which the first nucleic acid encodes is immunologicallycross reactive with the polypeptide encoded by the second nucleic acid.

(e) (ii) The terms “substantial identity” in the context of peptideindicates that a peptide comprises a sequence with at least 70% sequenceidentity to a reference sequence, preferably 80%, more preferably 85%,most preferably at least 90% or 95% sequence identity to the referencesequence over a specified comparison window. Preferably, optimalalignment is conducted using the homology alignment algorithm ofNeedleman et al. (1970) J. Mol. Biol. 48:443. An indication that twopeptide sequences are substantially identical is that one peptide isimmunologically reactive with antibodies raised against the secondpeptide. Thus, a peptide is substantially identical to a second peptide,for example, where the two peptides differ only by a conservativesubstitution.

Fragments and variants of the disclosed nucleotide sequences andproteins encoded thereby are also encompassed by the present invention.By “fragment” is intended a portion of the nucleotide sequence or aportion of the amino acid sequence and hence protein encoded thereby.Fragments of a nucleotide sequence may encode protein fragments thatretain the biological activity of the native protein and hence conferresistance to nematodes. Alternatively, fragments of a nucleotidesequence that are useful as hybridization probes generally do not encodefragment proteins retaining biological activity. Thus, fragments of anucleotide sequence may range from at least about 20 nucleotides, about50 nucleotides, about 100 nucleotides, and up to the entire nucleotidesequence encoding the proteins of the invention.

By “variants” is intended substantially similar sequences. Fornucleotide sequences, conservative variants include those sequencesthat, because of the degeneracy of the genetic code, encode the aminoacid sequence of one of the proteins conferring resistance to nematodes.Generally, nucleotide sequence variants of the invention will have atleast 70%, generally, 80%, preferably up to 90% sequence identity to itsrespective native nucleotide sequence.

By “variant” protein is intended a protein derived from the nativeprotein by deletion (so-called truncation) or addition of one or moreamino acids to the N-terminal and/or C-terminal end of the nativeprotein; deletion or addition of one or more amino acids at one or moresites in the native protein; or substitution of one or more amino acidsat one or more sites in the native protein. Such variants may resultfrom, for example, genetic polymorphism or from human manipulation.Methods for such manipulations are generally known in the art.

The proteins of the invention may be altered in various ways includingamino acid substitutions, deletions, truncations, and insertions.Methods for such manipulations are generally known in the art. Forexample, amino acid sequence variants of the activator proteins can beprepared by mutations in the DNA. Methods for mutagenesis and nucleotidesequence alterations are well known in the art. See, for example, Kunkelet al. (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al.(1987) Methods in Enzymol. 154:367-382; U.S. Pat. No. 4,873,192; Walkerand Gaastra (eds.) Techniques in Molecular Biology, MacMillan PublishingCompany, NY (1983) and the references cited therein. Thus, the genes andnucleotide sequences of the invention include both the naturallyoccurring sequences as well as variant and mutant forms. Likewise, theproteins of the invention encompass both naturally occurring proteins aswell as variants and modified forms thereof. Such variants will continueto possess the desired PARP activity. Obviously, the mutations that willbe made in the DNA encoding the variant must not place the sequence outof reading frame and preferably will not create complementary regionsthat could produce secondary mRNA structure. See, EP Patent ApplicationPublication No. 75,444.

PARP is present in all higher eukaryotes. Therefore, it is recognizedthat the nucleotide sequence encoding the PARP may be utilized from anyeukaryotic source, including vertebrates, arthropods, mollusks, slimemoulds, dinoflagellates, fungi, mammals, chicken, Xenopus and insects.See, for example, Heller et al. (1995) J. Biol. Chem. 270:11178-11180;Schreiber et al. (1995) Proc. Natl. Acad. Sci. USA 92:4753-4757; Ueda etal. (1985) Ann. Rev. Biochem. 54:73-100; Brightwell et al. (1975)Biochem. J. 147:119-129; Kofler et al. (1993) ibid 293:275-281; Collingeet al. (1994) Mol. Gen. Genet. 245:686-693; Scovassi et al. (1986) Eur.J. Biochem. 159:77-84; Simonin et al. (1991) Anal. Biochem. 195:226-231;Masutani et al. (1994) Eur. J. Biochem. 220:607-614; herein incorporatedby reference.

It is recognized that the plant cell can be transformed with anucleotide sequence encoding PARP, a nucleotide sequence encoding aportion of PARP, preferably the C-terminal portion of PARP, as well aswith a nucleotide sequence encoding the antisense sequence for the PARPgene, or portions thereof. In this manner, the level of expression ofthe PARP in the plant cell can be modulated, i.e., increased ordecreased, respectively. Levels of expression of the sense or antisensesequence can be regulated by the promoter utilized to express the gene.

Promoters for the expression of genes in plant cells are known in theart. Promoters are available for constitutive, tissue specific,inducible, etc. Such promoters include, for example, 35S promoter, Meyeret al. (1997) J. Gen. Virol. 78:3147-3151; biotin carboxylase, Bas etal. (1997) Plant Mol. Biol. 35:539-550; oxidase, Lasserre et al. (1997)Mol. Gen. Genet 256:211-222; cab, Shiina et al. (1997) Plant Physiol.115:477-483; phospholipase, Xu et al. (1997) Plant Physiol. 115:387-395;farnesyltransferase, Zhou et al. (1997) Plant J. 12:921-930;plastocyanin, Helliwell et al. (1997) Plant J. 12:499-506; CVMVpromoter, Verdaquer et al. (1996) Plant Mol. Biol. 31:1129-1139; actin,An et al. (1996) Plant J. 10:107-121; heat shock, Prandl et al. (1996)Plant Mol. Biol. 31:157-162; ubiquitin, thionin, 35S, Holtorf et al.(1995) Plant Mol. Biol. 29:637-646; Callis et al. (1990) J. Biol. Chem.265:12486-12493; histone, Atanossova et al. (1992) Plant J. 2:291-300;rol C, Fladung et al. (1993) Plant Mol. Biol. 23:749-757; histone,Brignon et al. (1993) Plant J. 4:445-457; Lepetit et al. (1992) Mol.Gen. Genet. 231:276-285.

As indicated, recent studies on the mechanism of PARP suggestsinvolvement of the enzyme in regulation of DNA repair, recombination andreplication. The enzyme is rapidly activated by DNA and exhibits a highaffinity for naked single-stranded or double-stranded DNA. Anyperturbation in the cellular morphology and/or physiology that causes achange in chromatin conformation generally results in a rapid increasein PARP activity. PARP is an important modulator of the fate of DNAintroduced into a plant cell. Accordingly, plants transformed witheither a sense or antisense PARP nucleotide sequence may be utilized toincrease transformation frequency in plant cells. Therefore, the presentinvention provides for the regulation of the levels of PARP in the plantcell to determine its effect on plant transformation and gene targeting.

It is further recognized that because the enzyme plays a role incellular stress, it may be beneficial to increase the levels of theenzyme to prevent plant disease or pathogen attack. In this manner,constitutive or inducible promoters may be utilized. Constitutivepromoters include, for example, those disclosed in U.S. Pat. Nos.5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680;5,268,463; 5,608,142, herein incorporated by reference. Induciblepromoters are known in the art and include, for example, pathogeninducible promoters, such as promoters from pathogenesis-relatedproteins (PR proteins) which are induced following infection by apathogen; e.g., PR proteins, SAR proteins, beta-1,3-glucanase,chitinase, etc. See, for example, Redolfi et al. (1983) Neth. J. PlantPathol. 89:245-254; Uknes et al. (1992) The Plant Cell 4:645-656; andVan Loon (1985) Plant Mol. Virol. 4:111-116. Of interest are promotersthat are expressed locally at or near the site of pathogen infection.See, for example, Marineau et al. (1987) Plant Mol. Biol. 9:335-342;Matton et al. (1989) Molecular Plant-Microbe Interactions 2:325-331;Somsisch et al. (1986) Proc. Natl. Acad. Sci. USA 83:2427-2430; Somsischet al. (1988) Molecular and General Genetics 2:93-98; and Yang, Y (1996)Proc. Natl. Acad. Sci. USA 93:14972-14977. See also, Chen et al. (1996)Plant J. 10:955-966; Zhang et al. (1994) Proc. Natl. Acad. Sci. USA91:2507-2511; Warner et al. (1993) Plant J. 3:191-201; Siebertz et al.(1989) Plant Cell 1:961-968; and the references cited therein. Ofparticular interest is the inducible promoter for the maize PRms gene,whose expression is induced by the pathogen Fusarium moniliforme (see,for example, Cordero et al. (1992) Physiological and Molecular PlantPathology 41:189-200).

The PARP genes or antisense nucleotides of the invention can beintroduced into any plant. The genes or nucleotide sequences to beintroduced will be used in expression cassettes for expression in anyplant of interest.

Such expression cassettes will comprise a transcriptional initiationregion linked to the gene encoding the PARP gene or antisense nucleotideof interest. Such an expression cassette is provided with a plurality ofrestriction sites for insertion of the gene of interest to be under thetranscriptional regulation of the regulatory regions. The expressioncassette may additionally contain selectable marker genes.

The transcriptional initiation region, the promoter, may be native oranalogous or foreign or heterologous to the plant host. Additionally,the promoter may be the natural sequence or alternatively a syntheticsequence. By foreign is intended that the transcriptional initiationregion is not found in the native plant into which the transcriptionalinitiation region is introduced. As used herein a chimeric genecomprises a coding sequence operably linked to a transcriptioninitiation region that is heterologous to the coding sequence.

The transcriptional cassette will include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region, aDNA sequence of interest, and a transcriptional and translationaltermination region functional in plants. The termination region may benative with the transcriptional initiation region, may be native withthe DNA sequence of interest, or may be derived from another source.Convenient termination regions are available from the Ti-plasmid of A.tumefaciens, such as the octopine synthase and nopaline synthasetermination regions. See also, Guerineau et al. (1991) Mol. Gen. Genet.262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991)Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroeet al. (1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids Res.17:7891-7903; Joshi et al. (1987) Nucleic Acid Res. 15:9627-9639.

The genes of the invention are provided in expression cassettes forexpression in the plant of interest. The cassette will include 5′ and 3′regulatory sequences operably linked to the gene of interest. Thecassette may additionally contain at least one additional gene to becotransformed into the organism. Alternatively, the additional gene(s)can be provided on another expression cassette. Where appropriate, thegene(s) may be optimized for increased expression in the transformedplant. That is, the genes can be synthesized using plant preferredcodons for improved expression. Methods are available in the art forsynthesizing plant preferred genes. See, for example, U.S. Pat. Nos.5,380,831, 5,436,391, and Murray et al. (1989) Nucleic Acids Res.17:477-498, herein incorporated by reference.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other such well-characterized sequenceswhich may be deleterious to gene expression. The G-C content of thesequence may be adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Whenpossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures.

The expression cassettes may additionally contain 5′ leader sequences inthe expression cassette construct. Such leader sequences can act toenhance translation. Translation leaders are known in the art andinclude: picornavirus leaders, for example, EMCV leader(Encephalomyocarditis 5′ noncoding region) (Elroy-Stein et al. (1989)PNAS USA 86:6126-6130); potyvirus leaders, for example, TEV leader(Tobacco Etch Virus) (Allison et al. (1986); MDMV leader (Maize DwarfMosaic Virus); Virology 154:9-20), and human immunoglobulin heavy-chainbinding protein (BiP), (Macejak et al. (1991) Nature 353:90-94;untranslated leader from the coat protein mRNA of alfalfa mosaic virus(AMV RNA 4), (Jobling et al. (1987) Nature 325:622-625; tobacco mosaicvirus leader (TMV), (Gallie, D. R. et al. (1989) Molecular Biology ofRNA, pages 237-256; and maize chlorotic mottle virus leader (MCMV)(Lommel, S. A. et al. (1991) Virology 81:382-385). See also,Della-Cioppa et al. (1987) Plant Physiology 84:965-968. Other methodsknown to enhance translation can also be utilized, for example, introns,and the like.

In preparing the expression cassette, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardsthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g. transitions andtransversions, may be involved.

The genes of the present invention can be used to transform any plant.In this manner, genetically modified plants, plant cells, plant tissue,seed, and the like can be obtained. Transformation protocols may varydepending on the type of plant or plant cell, i.e. monocot or dicot,targeted for transformation. Suitable methods of transforming plantcells include microinjection (Crossway et al. (1986) Biotechniques4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci.USA 83:5602-5606, Agrobacterium mediated transformation (Hinchee et al.(1988) Biotechnology 6:915-921), direct gene transfer (Paszkowski et al.(1984) EMBO J. 3:2717-2722), and ballistic particle acceleration (see,for example, Sanford et al., U.S. Pat. No. 4,945,050; Tomes et al.Direct DNA Transfer into Intact Plant Cells via MicroprojectileBombardment In Gamborg and Phillips (eds.) Plant Cell, Tissue and OrganCulture: Fundamental Methods, Springer-Verlag, Berlin (1995); and McCabeet al. (1988) Biotechnology 6:923-926). Also see, Weissinger et al.(1988) Ann. Rev. Genet. 22:421-477; Sanford et al. (1987) ParticulateScience and Technology 5:27-37 (onion); Christou et al. (1988) PlantPhysiol. 87:671-674 (soybean); McCabe et al. (1988) Bio/Technology6:923-926 (soybean); Datta et al. (1990) Biotechnology 8:736-740 (rice);Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize);Klein et al. (1988) Biotechnology 6:559-563 (maize); Tomes et al. DirectDNA Transfer into Intact Plant Cells via Microprojectile Bombardment inGamborg and Phillips (eds.) Plant Cell, Tissue and Organ Culture:Fundamental Methods, Springer-Verlag, Berlin (1995) (maize); Klein etal. (1988) Plant Physiol. 91:440-444 (maize); Fromm et al. (1990)Biotechnology 8:833-839 (maize); Hooydaas-Van Slogteren & Hooykaas(1984) Nature (London) 311:763-764; Bytebier et al. (1987) Proc. Natl.Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet et al. (1985) in TheExperimental Manipulation of Ovule Tissues ed. G. P. Chapman et al., pp.197-209. Longman, N.Y. (pollen); Kaeppler et al. (1990) Plant CellReports 9:415-418; and Kaeppler et al. (1992) Theor. Appl. Genet.84:560-566 (whisker-mediated transformation); D Halluin et al. (1992)Plant Cell 4:1495-1505 (electroporation); Li et al. (1993) Plant CellReports 12:250-255 and Christou and Ford (1995) Annals of Botany75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750(maize via Agrobacterium tumefaciens); all of which are hereinincorporated by reference.

The cells which have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting hybrid having the desired phenotypic characteristicidentified. Two or more generations may be grown to ensure that thesubject phenotypic characteristic is stably maintained and inherited andthen seeds harvested to ensure the desired phenotype or other propertyhas been achieved.

The present invention also provides isolated nucleic acid comprisingpolynucleotides of sufficient length and complementarity to a gene touse as probes or amplification primers in the detection, quantitation,or isolation of gene transcripts. For example, isolated nucleic acids ofthe present invention can be used as probes in detecting deficiencies inthe level of mRNA in screenings for desired transgenic plants, fordetecting mutations in the gene (e.g., substitutions, deletions, oradditions), for monitoring upregulation of expression or changes inenzyme activity in screening assays of compounds, for detection of anynumber of allelic variants (polymorphisms) of the gene, or for use asmolecular markers in plant breeding programs. The isolated nucleic acidsof the present invention can also be employed for use in sense orantisense suppression of a PARP gene in a host cell, tissue, or plant.See, Tools to Determine the Function of Genes, 1995 Proceedings of theFiftieth Annual Corn and Sorghum Industry Research Conference, AmericanSeed Trade Association, Washington, D.C., 1995. Additionally,non-translated 5 or 3 regions of the polynucleotides of the presentinvention can be used to modulate turnover of heterologous mRNAs and/orprotein synthesis.

The present invention provides a method of genotyping a plant comprisinga polynucleotide of the present invention. Preferably, the plant is amonocot, such as maize or sorghum. Genotyping provides a means ofdistinguishing homologs of a chromosome pair and can be used todifferentiate segregants in a plant population. Molecular marker methodscan be used for phylogenetic studies, characterizing geneticrelationships among crop varieties, identifying crosses or somatichybrids, localizing chromosomal segments affecting monogenic traits, mapbased cloning, and the study of quantitative inheritance. See, e.g.,Plant Molecular Biology: A Laboratory Manual, Chapter 7, Clark, Ed.,Springer-Verlag, Berlin (1997). For molecular marker methods, seegenerally, The DNA Revolution by Andrew H. Paterson 1996 (Chapter 2) in:Genome Mapping in Plants (ed. Andrew H. Paterson) by Academic Press/R.G. Landis Company, Austin, Tx., pp.7-21.

The particular method of genotyping in the present invention may employany number of molecular marker analytic techniques such as, but notlimited to, restriction fragment length polymorphisms (RFLPs). RFLPs arethe product of allelic differences between DNA restriction fragmentscaused by nucleotide sequence variability. As is well known to those ofskill in the art, RFLPs are typically detected by extraction of genomicDNA and digestion with a restriction enzyme. Generally, the resultingfragments are separated according to size and hybridized with a probe;single copy probes are preferred. Restriction fragments from homologouschromosomes are revealed. Differences in fragment size among allelesrepresent an RFLP. Thus, the present invention further provides a meansto follow segregation of a PARP gene or nucleic acid of the presentinvention as well as chromosomal sequences genetically linked to thesegenes or nucleic acids using such techniques as RFLP analysis. Linkedchromosomal sequences are within 50 centiMorgans (cM), often within 40or 30 cM, preferably within 20 or 10 cM, more preferably within 5, 3, 2,or 1 cM of a PARP gene.

In the present invention, the nucleic acid probes employed for molecularmarker mapping of plant nuclear genomes selectively hybridize, underselective hybridization conditions, to a gene encoding a polynucleotideof the present invention. In preferred embodiments, the probes areselected from polynucleotides of the present invention. Typically, theseprobes are cDNA probes or Pst I genomic clones. The length of the probesis discussed in greater detail, supra, but are typically at least 15bases in length, more preferably at least 20, 25, 30, 35, 40, or 50bases in length. Generally, however, the probes are less than about 1kilobase in length. Preferably, the probes are single copy probes thathybridize to a unique locus in a haploid chromosome complement.

The method of detecting an RFLP comprises the steps of (a) digestinggenomic DNA of a plant with a restriction enzyme; (b) hybridizing anucleic acid probe, under selective hybridization conditions, to asequence of a polynucleotide of the present of said genomic DNA; (c)detecting therefrom a RFLP. Other methods of differentiating polymorphic(allelic) variants of polynucleotides of the present invention can behad by utilizing molecular marker techniques well known to those ofskill in the art including such techniques as: 1) single strandedconformation analysis (SSCP); 2) denaturing gradient gel electrophoresis(DGGE); 3) RNase protection assays; 4) allele-specific oligonucleotides(ASOs); 5) the use of proteins which recognize nucleotide mismatches,such as the E. coli mutS protein; and 6) allele-specific PCR. Otherapproaches based on the detection of mismatches between the twocomplementary DNA strands include clamped denaturing gel electrophoresis(CDGE); heteroduplex analysis (HA); and chemical mismatch cleavage(CMC). Thus, the present invention further provides a method ofgenotyping comprising the steps of contacting, under stringenthybridization conditions, a sample suspected of comprising apolynucleotide of the present invention with a nucleic acid probe.Generally, the sample is a plant sample; preferably, a sample suspectedof comprising a maize polynucleotide of the present invention (e.g.,gene, mRNA). The nucleic acid probe selectively hybridizes, understringent conditions, to a subsequence of a polynucleotide of thepresent invention comprising a polymorphic marker. Selectivehybridization of the nucleic acid probe to the polymorphic markernucleic acid sequence yields a hybridization complex. Detection of thehybridization complex indicates the presence of that polymorphic markerin the sample. In preferred embodiments, the nucleic acid probecomprises a polynucleotide of the present invention.

EXPERIMENTAL Materials and Methods

Chemicals and Reagents

All chemicals used in this study were of molecular biology grade. Trizmabase (Tris(hydroxymethyl)aminomethane; abbreviated hereafter as Tris),N-2 hydroxy-ethyl-piperazine-N′-2-ethane sulfonic acid (Hepes),Ethylenediaminetetraacetic acid, sodium salt (EDTA), Magnesium chloride,Urea, were procured from Sigma Chemical Co. Analytical grade glycerolwas obtained from Baxter. Dithiothreitol (DTT), PefablocSC, Pepstatin,Bestatin, all restriction enzymes, DNA and RNA purification kits as wellas markers were purchased from Boehringer Mannheim. Immunodetection kitsfor Western blots, silver staining and Colloidal Coomassie Blue stainingwere from Novex. All radioactive chemicals were purchased fromNEN-Dupont and NEN. Chromotopographic resins were purchased either fromSigma, BioRad or Pharmacia.

Cell Culture

The enzyme is isolated from a Hi II embryogenic callus cell line.Exponentially growing cultures of 612B4 cells were maintained in dark at28° C. (Armstrong et al. (1992) Theor. Appl. Genet. 84:755-762). Thecell suspensions are in MS medium supplemented with2-4-dichlorophenoxyaxcetic acid (2.5 mg/l). Cultures are grown for aweek on a gyroratatory shaker at 150 rpm and harvested by decantation.Routinely, 60-80 g of cells are obtained from 800-900 ml cultures grownin 12-14 flasks.

Cells are harvested by filtration and used to prepare whole cellextracts (WCE) form 612B4 cells using the Bionebulizer (Glas-Col, TerreHaute, Ind.). The process for WCE preparation is outlined in Schema 1.All operations were carried out at 4° C. or on ice unless mentionedotherwise.

Chromatography on Heparin-agarose: About 300 ml of Heparin-agarose(Sigma) was washed extensively with 20 mM Hepes-KOH pH 7.9, 0.1 mM EDTA,20% glycerol (HGED buffer), packed into a 5.0×30 cm Econo column(Biorad) and connected to the Econo System (Biorad). The matrix wasequilibrated with HGED+100 mM KCI. Three batches of crude WCE extract(approx. 1.8-2.0 g of total pooled protein in 60-80 ml) were loaded onthe column at a flow rate of 15-20 ml/hr. The column was washedextensively with equilibration buffer till the A₂₈₀ of the effluentwas<0.1 unit (approx. 900 ml).

Small aliquots of peak fractions were saved for PARP assays and allfractions (7-8 ml each) showing A₂₈₀>0.1 unit were pooled. Protein wasprecipitated by adding solid ammonium sulfate (0.4 g/ml). The mixturewas centrifuged at 40,000×g for 30 min., dissolved in minimum amountHGED and dialyzed against HGED+100 mM KCI containing Pefabloc and DTT.This fraction is designated HA-1. The column was further washed with 900ml each HGED+400 mM KCI followed by HGED+1 M KCI. Fractions from bothwashes were processed as above and designated as HA-2 and HA-3. PARPassays were performed on HA-1, 2 and 3 and the active fraction (HA-2)was used for further purification.

Chromatography on DNA-cellulose: DNA-cellulose (Sigma) was washedextensively with HGED and packed in the Econo column (2.5×30 cm). Thecolumn was connected to the Econo System and equilibrated with HGED+100mM KCI. Partially pure PARP from three Heparin-agarose column was loadedon the DNA-cellulose column. Unbound protein was removed by washing withHGED+100 mM KCI (200 ml; designated as DC-1). The bound protein waseluted with HGED+1M KCI (designated DC-2). All fractions was processedas described above for activity and protein.

Chromatography on Histone-agarose: Histone-agarose (Sigma) was washedextensively with HGED and packed into an Econo column (1.5×15 cm). Thecolumn was equilibrated in HGED+100 mM KCI. Active fraction fromDNA-cellulose (DC-2) was further fractionated on Histone-agarose bywashing the column successively with HGED containing 100 mM, 400 mM and1 M KCI. All fractions were processed as above and dialyzed against 20mM Tris-HCI buffer pH 7.9 containing 100 mM KCI.

Chromatography on Mini-Q column: Mini-Q column (Pharmacia) was connectedto Smart-LC (Pharmacia) and equilibrated by washing with five bedvolumes of HGED followed by five bed volumes of Tris-HCI buffer+100 mMKCI. Active PARP from the Histone-agarose step was loaded on the column.The column was washed with three bed volumes of equilibration buffer and400 μl fractions were collected. Column was further developed using astep gradient of KCI at 400 mM, 600 mM and 1M in Tris-HCI, pH 8.0. Allfractions were tested for PARP activity as described below.

Enzyme Assays

Catalytic activity of PARP is assayed following published protocols(Shah et al. (1995) Anal. Biochem 227:1-13) with modifications suitablefor the plant enzyme. Briefly, the enzyme (in a total volume of 25 μl of20 mM Hepes pH 7.9, 100 mM KCI) is incubated with 2.5-5 μCi of∞-³²P-NAD+, 2 μl/ml final concentration of bovine histone (fraction IV),2 μg of activated calf thymus DNA and 0.5 mM DTT. The reactions arecarried out at 6° C. unless otherwise mentioned. At the end of theappropriate time intervals, the labeled protein is precipitated with 25%TCA. The precipitate is collected by centrifugation at 16,000×g for 10min., washed 2× with 5% TCA and counted in a LSC. Protein heated at 65°C. for 5 min. is used as a negative control.

Microsequencing

Protein samples obtained from the Mini-Q column purification step waselectrophoresed in duplicate on a 10% polyacrylamide gels using 0.1% SDSin the running buffer (Shah et al. (1995) Anal. Biochem 227:1-13). Onehalf of the gel was used to detect protein bands with a ColloidalCoormassie staining kit (Novex) following manufacturer s instructions.The other half was used in the activity blot assay to confirm positionof the active PARP on the gel. Stained protein band corresponding toactive PARP was cut out from the gel and used microsequencing carriedout at the W. M. Keck Foundation Biotechnology Resource Laboratory ifYale University. In gel tryptic digestion of the protein, MatrixAssisted Laser Desorption Mass Spectrometry (MALDI-MS) of the isolatedpeptides, and amino sequencing of representative peptides was carriedout following protocols detailed elsewhere (Stone et al. (1990) In:Methods in Enzyomology 193:389-412); (Stone et al. (1991) In: Methods inProtein Sequence Anal.ysis 133-141); Williams et al. (1995) In:Techniques in Protein Chemistry 6:143-152); Williams et al. (1995) In:Protein Protocol Handbook 365-378).

Antipeptide Antibodies

Synthesis of the peptide antigens and antibody generation was carriedout at Research Genetics, Inc. Two peptides (P-1 and P-2) were used asfor antibody generation using two different protocols. In the firstprotocol, peptide P-1 was synthesized as a multiple antigenic peptide(MAP) following published protocols (Tam, J. P. (1988) Proc. Nat. Acad.Sci. USA 85:5409-5413). Antiserum was collected and analyzed forcross-reactivity to PARP using Western blots (Shah et al. (1995) Anal.Biochem 227:1-13). In the second protocol, P-2 was synthesized as MAP(Tam, J. P. (1988) Proc. Natl. Acad. Sci. USA 85:5409-5413) as well as alinear peptide (Barany et al. (1980) In: The Peptides 2:1-284). Thelinear peptide was conjugated to hemocyanin using published methods(Walter et al. (1980) Proc. Natl. Acad. Sci. USA 77:5197-5200) and usedfor immunization. The immunization protocol for both types of antigenswas essentially the same and is detailed below.

Two New Zealand rabbits (4-6 months old) were used for immunization witheach type of antigen. The antigens were prepared by dissolving 500 μgMAP peptide in 500 μl of saline and mixed with equal 500 μl of completeFreund's adjuvant and injected subcutaneously at three to four dorsalsites. Same concentration of each antigen (in saline) was mixed withequal volume of incomplete Freund's adjuvant and injected as before attwo, four and six weeks after the first immunization. Animals were bledfrom the auricular artery to collect 30-50 ml blood on days 0, 27, 57and 69. Blood samples were allowed to clot at room temperature for 15min. and serum was isolated from each sample by centrifugation at5,000×g for 10 min. Cell-free serum was decanted gently into a cleantube and stored at −20° C. till further use.

cDNA Cloning

Total RNA was isolated from corn tissues with TRIzol Reagent (LifeTechnology, Inc. Gaithersburg, Md.) using a modification of theguanidine isothiocyanate/acid-phenol procedure described by Chomczynskiand Sacchi (Chomczynski et al. (1987) Anal. Biochem 162:156). In brief,plant tissue samples were pulverized in liquid nitrogen before theaddition of the TRIzol Reagent, and then were further homogenized with amortar and pestle. Addition of chloroform by centrifugation wasconducted for separation of an aqueous phase and an organic phase. Thetotal RNA was recovered by precipitation with isopropyl alcohol from theaqueous phase.

The selection of poly(A)+RNA from total RNA was performed usingPolyATract system (Promega Corporation, Madison, Wis.). In brief,biotinylated oligo (dT) primers were used to hybridize to the 3′ poly(A)tails on mRNA. The hybrids were captured using streptavidin coupled toparamagnetic particles and a magnetic separation stand. The mRNA waswashed at high stringent condition and eluted by RNase-free deionizedwater.

Synthesis of the cDNA was performed and unidirectional cDNA librarieswere constructed using the SuperScript Plasmid System (Life Technology,Inc., Gaithersburg, Md.). First strand of cDNA was synthesized bypriming an oligo(dT) primer containing a Not I site. The reaction wascatalyzed by SuperScript Reverse Transcriptase II at 45° C. The secondstrand of cDNA was labeled with alpha-³²P-dCTP and portions of themolecules smaller than 500 base pairs and unligated adapters wereremoved by Sephacryl-S400 chromatography. The selected cDNA moleculeswere ligated into pSPORT1 reference vector between the Not I and Sal Isites.

Individual colonies were picked and DNA was prepared either by PCR withM13 forward primers and M13 reverse primers, or by plasmid miniprepisolation. All the cDNA clones were sequenced using M13 reverse primers.

Analytical

Protein was estimated by the Bradford method (Bradford, M. (1976) ibid72:248-254) using bovine γ-globulin as standard. Activity blots, Westernblots and product analysis were performed essentially followingpublished protocols (10, 20-22), except that all essays were carried outat 6° C.

Identification of Zinc Fingers

Two PCR primers were designed to encompass both the Zinc fingers of themaize PARP sequence. These primers were used for reverse transcriptaseassisted PCR using the Titan 1 tube RT-PCR kit from BoeheringerMannheim. Maize callus and leaf mRNA was used as template. The PCRproduct was purified using Qia Quick PCR product purification columns(Qiagen) and sequenced using an ABI sequencer. Sequenced data is shownin SEQ ID NO. 5.

Isolation of PARP from maize cells Scheme I Cells ↓ (60-80 g) ↓ Suspendin Nebulization Buffer (4 ml/g) ↓ (20 mM Hepes pH 7.9, 20% glycerol, 0.4molal sorbitol, 0.1 mM EDTA) (+DTT and PI cocktail) ↓ Bionebulization at100 psi × 4 ↓ Filter through cheesecloth ↓ Filtrate ↓ Add 0.1 volume ofSASS ↓ Mix gently for 1 hr. ↓ Centrifuge at 100,000 × g ↓ Add 0.4 ofsolid (NH₄)₂SO₄ per ml/S100 ↓ Mix for 30 min. ↓ Centrifuge at 40,000 × gfor 30 min. ↓ Dissolve precipitate in HGED buffer and dialyze overnight↓ Centrifuge at 40,000 × g for 30 min. ↓ Store WCE at −80° C.

ll publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 5 <210> SEQ ID NO 1 <211>LENGTH: 2949 <212> TYPE: DNA <213> ORGANISM: Zea mays <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (1)...(2949) <221> NAME/KEY:misc_feature <222> LOCATION: 1584, 1588, 2078, 2107 <223> OTHERINFORMATION: n = A,T,C or G <400> SEQUENCE: 1 atg gcg gcg ccg cca aaggcg tgg aag gcg gag tat gcc aag tct ggg 48 Met Ala Ala Pro Pro Lys AlaTrp Lys Ala Glu Tyr Ala Lys Ser Gly 1 5 10 15 cgg gcc tcg tgc aag tcatgc cgg tcc cct atc gcc aag gac cag ctc 96 Arg Ala Ser Cys Lys Ser CysArg Ser Pro Ile Ala Lys Asp Gln Leu 20 25 30 cgt ctt ggc aag atg gtt caggcg tca cag ttc gac ggc ttc atg ccg 144 Arg Leu Gly Lys Met Val Gln AlaSer Gln Phe Asp Gly Phe Met Pro 35 40 45 atg tgg aac cat gcc agg tgc atcttc agc aag aag aac cag ata aaa 192 Met Trp Asn His Ala Arg Cys Ile PheSer Lys Lys Asn Gln Ile Lys 50 55 60 tcc gtt gac gat gtt gaa ggg ata gatgca ctt aga tgg gat gat caa 240 Ser Val Asp Asp Val Glu Gly Ile Asp AlaLeu Arg Trp Asp Asp Gln 65 70 75 80 gag aag ata cga aac tac gtt ggg agtgcc tca gct ggt aca agt tct 288 Glu Lys Ile Arg Asn Tyr Val Gly Ser AlaSer Ala Gly Thr Ser Ser 85 90 95 aca gct gct cct cct gag aaa tgt aca attgag att gct cca tct gcc 336 Thr Ala Ala Pro Pro Glu Lys Cys Thr Ile GluIle Ala Pro Ser Ala 100 105 110 cgt act tca tgt aga cga tgc agt gaa aagatt aca aaa gga tcg gtc 384 Arg Thr Ser Cys Arg Arg Cys Ser Glu Lys IleThr Lys Gly Ser Val 115 120 125 cgt ctt tca gct aag ctt gag agt gaa ggtccc aag ggt ata cca tgg 432 Arg Leu Ser Ala Lys Leu Glu Ser Glu Gly ProLys Gly Ile Pro Trp 130 135 140 tat cat gcc aac tgt ttc ttt gag gta tccccg tct gca act gtt gag 480 Tyr His Ala Asn Cys Phe Phe Glu Val Ser ProSer Ala Thr Val Glu 145 150 155 160 aag ttc tca ggc tgg gat act ttg tccgat gag gat aag aga acc atg 528 Lys Phe Ser Gly Trp Asp Thr Leu Ser AspGlu Asp Lys Arg Thr Met 165 170 175 ctc gat ctt gtt aaa aaa gat gtt ggcaac aat gaa caa aat aag ggt 576 Leu Asp Leu Val Lys Lys Asp Val Gly AsnAsn Glu Gln Asn Lys Gly 180 185 190 tcc aag cgc aag aaa agt gaa aat gatatt gat agc tac aaa tcc gcc 624 Ser Lys Arg Lys Lys Ser Glu Asn Asp IleAsp Ser Tyr Lys Ser Ala 195 200 205 agg tta gat gaa agt aca tct gaa ggtaca gtg cga aac aaa ggg caa 672 Arg Leu Asp Glu Ser Thr Ser Glu Gly ThrVal Arg Asn Lys Gly Gln 210 215 220 ctt gta gac cca cgt ggt tcc aat actagt tca gct gat atc caa cta 720 Leu Val Asp Pro Arg Gly Ser Asn Thr SerSer Ala Asp Ile Gln Leu 225 230 235 240 aag ctt aag gag caa agt gac acactt tgg aag tta aag gat gga ctt 768 Lys Leu Lys Glu Gln Ser Asp Thr LeuTrp Lys Leu Lys Asp Gly Leu 245 250 255 aag act cat gta tcg gct gct gaatta agg gat atg ctt gag gct aat 816 Lys Thr His Val Ser Ala Ala Glu LeuArg Asp Met Leu Glu Ala Asn 260 265 270 ggg cag gat aca tca gga cca gaaagg cac cta ttg gat cgc tgt gcg 864 Gly Gln Asp Thr Ser Gly Pro Glu ArgHis Leu Leu Asp Arg Cys Ala 275 280 285 gat gga atg cta ttt gga gcg ctgggt cct tgc cca gtc tgt gct aat 912 Asp Gly Met Leu Phe Gly Ala Leu GlyPro Cys Pro Val Cys Ala Asn 290 295 300 ggc atg tac tat tat aat ggt cagtac caa tgc agt ggt aat gtg tca 960 Gly Met Tyr Tyr Tyr Asn Gly Gln TyrGln Cys Ser Gly Asn Val Ser 305 310 315 320 gag tgg tcc aag tgt aca tactct gcc aca gaa cct gtc cgc gtt aag 1008 Glu Trp Ser Lys Cys Thr Tyr SerAla Thr Glu Pro Val Arg Val Lys 325 330 335 aag aag tgg caa att cca catgga aca aag aat gat tac ctt atg aag 1056 Lys Lys Trp Gln Ile Pro His GlyThr Lys Asn Asp Tyr Leu Met Lys 340 345 350 tgg ttc aaa tct caa aag gttaag aaa cca gag agg gtt ctt cca cca 1104 Trp Phe Lys Ser Gln Lys Val LysLys Pro Glu Arg Val Leu Pro Pro 355 360 365 atg tca cct gag aaa tct ggaagt aaa gca act cag aga aca tca ttg 1152 Met Ser Pro Glu Lys Ser Gly SerLys Ala Thr Gln Arg Thr Ser Leu 370 375 380 ctg tct tct aaa ggg ttg gataaa tta agg ttt tct gtt gta gga caa 1200 Leu Ser Ser Lys Gly Leu Asp LysLeu Arg Phe Ser Val Val Gly Gln 385 390 395 400 tca aaa gaa gca gca aatgag tgg att gag aag ctc aaa ctt gct ggt 1248 Ser Lys Glu Ala Ala Asn GluTrp Ile Glu Lys Leu Lys Leu Ala Gly 405 410 415 gcc aac ttc tat gcc agggtt gtc aaa gat att gat tgt tta att gca 1296 Ala Asn Phe Tyr Ala Arg ValVal Lys Asp Ile Asp Cys Leu Ile Ala 420 425 430 tgt ggt gag ctc gac aatgaa aat gct gaa gtc agg aaa gca agg agg 1344 Cys Gly Glu Leu Asp Asn GluAsn Ala Glu Val Arg Lys Ala Arg Arg 435 440 445 ctg aag ata cca att gtaagg gag ggt tac att gga gaa tgt gtt aaa 1392 Leu Lys Ile Pro Ile Val ArgGlu Gly Tyr Ile Gly Glu Cys Val Lys 450 455 460 aga aca aaa tgc tgc catttg att tgt ata aac tgg aat gcc tta gag 1440 Arg Thr Lys Cys Cys His LeuIle Cys Ile Asn Trp Asn Ala Leu Glu 465 470 475 480 tcc tca aaa ggc mgtact gtc act gtt aaa gtt aag ggc cga agt gct 1488 Ser Ser Lys Gly Xaa ThrVal Thr Val Lys Val Lys Gly Arg Ser Ala 485 490 495 tgt tca tya agt cctcyg gtt tgc aag aat act gct cac att cct tra 1536 Cys Ser Xaa Ser Pro XaaVal Cys Lys Asn Thr Ala His Ile Pro Xaa 500 505 510 gra tgg gaa aag catata caa tgc amc ctt aaa cat gtt ctg acc tgn 1584 Xaa Trp Glu Lys His IleGln Cys Xaa Leu Lys His Val Leu Thr Xaa 515 520 525 cac nag gtg tgy acaggc tac tat gta ctc cag atc att gaa cag gat 1632 His Xaa Val Cys Thr GlyTyr Tyr Val Leu Gln Ile Ile Glu Gln Asp 530 535 540 gat ggg tct gag tgctac gta ttt cgt aag tgg gga cgg gtt ggg agt 1680 Asp Gly Ser Glu Cys TyrVal Phe Arg Lys Trp Gly Arg Val Gly Ser 545 550 555 560 gag aaa att ggaggg caa aaa ctg gag gag atg tca aaa act gag gca 1728 Glu Lys Ile Gly GlyGln Lys Leu Glu Glu Met Ser Lys Thr Glu Ala 565 570 575 atc aag gaa ttcaaa aga tta ttt ctt gag aag act gga aac tca tgg 1776 Ile Lys Glu Phe LysArg Leu Phe Leu Glu Lys Thr Gly Asn Ser Trp 580 585 590 gaa gct tgg gaatgt aaa acc aat ttt cgg aag cag cct ggg aga ttt 1824 Glu Ala Trp Glu CysLys Thr Asn Phe Arg Lys Gln Pro Gly Arg Phe 595 600 605 tac cca ctt gatgtt gat tat ggt gtt aag aaa gca cca aaa cgg aaa 1872 Tyr Pro Leu Asp ValAsp Tyr Gly Val Lys Lys Ala Pro Lys Arg Lys 610 615 620 gat atc agt gaaatg aaa agt tct ctt gct cct caa ttg cta gaa ctc 1920 Asp Ile Ser Glu MetLys Ser Ser Leu Ala Pro Gln Leu Leu Glu Leu 625 630 635 640 atg aag atgctt ttc aat gtg gag aca tat aga gct gct atg atg gaa 1968 Met Lys Met LeuPhe Asn Val Glu Thr Tyr Arg Ala Ala Met Met Glu 645 650 655 ttt gaa awtaat atg tca gaa atg cct ctt ggg aag cta agc mag gra 2016 Phe Glu Xaa AsnMet Ser Glu Met Pro Leu Gly Lys Leu Ser Xaa Xaa 660 665 670 aat att gagraa gga ttt gaa gca tta act krg rta cmg rat tta ttt 2064 Asn Ile Glu XaaGly Phe Glu Ala Leu Thr Xaa Xaa Xaa Xaa Leu Phe 675 680 685 gaa gga caccgc tna tca agc act ggc ttg ttr gag aaa gct naa ttg 2112 Glu Gly His ArgXaa Ser Ser Thr Gly Leu Xaa Glu Lys Ala Xaa Leu 690 695 700 ttg ytg sgagcm ats syt ttt tca ctc tta tcc ctt cta ttc atc ctc 2160 Leu Xaa Xaa XaaXaa Xaa Phe Ser Leu Leu Ser Leu Leu Phe Ile Leu 705 710 715 720 ata ttatac ggg atg agg atg att tca tat tca aag gcg aaa atg ctt 2208 Ile Leu TyrGly Met Arg Met Ile Ser Tyr Ser Lys Ala Lys Met Leu 725 730 735 gaa gctctg cag gat att gaa att gct tca aag ata gtt ggc ttc gat 2256 Glu Ala LeuGln Asp Ile Glu Ile Ala Ser Lys Ile Val Gly Phe Asp 740 745 750 agc gacagt gat gaa tct ctt gat gat aaa tat atg aaa ctt cac tgt 2304 Ser Asp SerAsp Glu Ser Leu Asp Asp Lys Tyr Met Lys Leu His Cys 755 760 765 gac atcacc ccg ctg gct cac gat agt gaa gat tac aag tta att gag 2352 Asp Ile ThrPro Leu Ala His Asp Ser Glu Asp Tyr Lys Leu Ile Glu 770 775 780 cag tatctc ctc aac aca cat gct cct act cac aag gac tgg tcg ctg 2400 Gln Tyr LeuLeu Asn Thr His Ala Pro Thr His Lys Asp Trp Ser Leu 785 790 795 800 gaactg gag gaa gtt ttt tca ctt gat cga gat gga gaa ctt aat aag 2448 Glu LeuGlu Glu Val Phe Ser Leu Asp Arg Asp Gly Glu Leu Asn Lys 805 810 815 tactca aga tat aaa aat aat ctg cat aac aag atg cta tta tgg cac 2496 Tyr SerArg Tyr Lys Asn Asn Leu His Asn Lys Met Leu Leu Trp His 820 825 830 ggttca agg ttg acg aat ttt gtg gga att ctt agt caa ggg cta aga 2544 Gly SerArg Leu Thr Asn Phe Val Gly Ile Leu Ser Gln Gly Leu Arg 835 840 845 attgca cct cct gag gca cct gtt act ggc tat atg ttc ggc aaa ggc 2592 Ile AlaPro Pro Glu Ala Pro Val Thr Gly Tyr Met Phe Gly Lys Gly 850 855 860 ctctac ttt gca gat cta gta agc aag agc gca caa tac tgt tat gtg 2640 Leu TyrPhe Ala Asp Leu Val Ser Lys Ser Ala Gln Tyr Cys Tyr Val 865 870 875 880gat agg aat aat cct gta ggt ttg atg ctt ctt tct gag gtt gct tta 2688 AspArg Asn Asn Pro Val Gly Leu Met Leu Leu Ser Glu Val Ala Leu 885 890 895gga gac atg tat gaa cta aag aaa gcc acg tcc atg gac aaa cct cca 2736 GlyAsp Met Tyr Glu Leu Lys Lys Ala Thr Ser Met Asp Lys Pro Pro 900 905 910aga ggg aag cat tcg acc aag gga tta ggc aaa acc gtg cca ctg gag 2784 ArgGly Lys His Ser Thr Lys Gly Leu Gly Lys Thr Val Pro Leu Glu 915 920 925tca gag ttt gtg aag tgg agg gat gat gtc gta gtt ccc tgc ggc aag 2832 SerGlu Phe Val Lys Trp Arg Asp Asp Val Val Val Pro Cys Gly Lys 930 935 940ccg gtg cca tca tca att agg agc tct gaa ctc atg tac aat gag tac 2880 ProVal Pro Ser Ser Ile Arg Ser Ser Glu Leu Met Tyr Asn Glu Tyr 945 950 955960 atc gtc tac aac aca tcc cag gtg aag atg cag ttc ttg ctg aag gtg 2928Ile Val Tyr Asn Thr Ser Gln Val Lys Met Gln Phe Leu Leu Lys Val 965 970975 cgt ttc cat cac aag agg tag 2949 Arg Phe His His Lys Arg * 980 <210>SEQ ID NO 2 <211> LENGTH: 982 <212> TYPE: PRT <213> ORGANISM: Zea mays<220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: 485, 499, 502,512, 513, 521, 528, 530, 659, 671, 672, 676, 683, 684, 685, 686, 693,699, 703, 706, 707, 708, 709, 710 <223> OTHER INFORMATION: Xaa = AnyAmino Acid <400> SEQUENCE: 2 Met Ala Ala Pro Pro Lys Ala Trp Lys Ala GluTyr Ala Lys Ser Gly 1 5 10 15 Arg Ala Ser Cys Lys Ser Cys Arg Ser ProIle Ala Lys Asp Gln Leu 20 25 30 Arg Leu Gly Lys Met Val Gln Ala Ser GlnPhe Asp Gly Phe Met Pro 35 40 45 Met Trp Asn His Ala Arg Cys Ile Phe SerLys Lys Asn Gln Ile Lys 50 55 60 Ser Val Asp Asp Val Glu Gly Ile Asp AlaLeu Arg Trp Asp Asp Gln 65 70 75 80 Glu Lys Ile Arg Asn Tyr Val Gly SerAla Ser Ala Gly Thr Ser Ser 85 90 95 Thr Ala Ala Pro Pro Glu Lys Cys ThrIle Glu Ile Ala Pro Ser Ala 100 105 110 Arg Thr Ser Cys Arg Arg Cys SerGlu Lys Ile Thr Lys Gly Ser Val 115 120 125 Arg Leu Ser Ala Lys Leu GluSer Glu Gly Pro Lys Gly Ile Pro Trp 130 135 140 Tyr His Ala Asn Cys PhePhe Glu Val Ser Pro Ser Ala Thr Val Glu 145 150 155 160 Lys Phe Ser GlyTrp Asp Thr Leu Ser Asp Glu Asp Lys Arg Thr Met 165 170 175 Leu Asp LeuVal Lys Lys Asp Val Gly Asn Asn Glu Gln Asn Lys Gly 180 185 190 Ser LysArg Lys Lys Ser Glu Asn Asp Ile Asp Ser Tyr Lys Ser Ala 195 200 205 ArgLeu Asp Glu Ser Thr Ser Glu Gly Thr Val Arg Asn Lys Gly Gln 210 215 220Leu Val Asp Pro Arg Gly Ser Asn Thr Ser Ser Ala Asp Ile Gln Leu 225 230235 240 Lys Leu Lys Glu Gln Ser Asp Thr Leu Trp Lys Leu Lys Asp Gly Leu245 250 255 Lys Thr His Val Ser Ala Ala Glu Leu Arg Asp Met Leu Glu AlaAsn 260 265 270 Gly Gln Asp Thr Ser Gly Pro Glu Arg His Leu Leu Asp ArgCys Ala 275 280 285 Asp Gly Met Leu Phe Gly Ala Leu Gly Pro Cys Pro ValCys Ala Asn 290 295 300 Gly Met Tyr Tyr Tyr Asn Gly Gln Tyr Gln Cys SerGly Asn Val Ser 305 310 315 320 Glu Trp Ser Lys Cys Thr Tyr Ser Ala ThrGlu Pro Val Arg Val Lys 325 330 335 Lys Lys Trp Gln Ile Pro His Gly ThrLys Asn Asp Tyr Leu Met Lys 340 345 350 Trp Phe Lys Ser Gln Lys Val LysLys Pro Glu Arg Val Leu Pro Pro 355 360 365 Met Ser Pro Glu Lys Ser GlySer Lys Ala Thr Gln Arg Thr Ser Leu 370 375 380 Leu Ser Ser Lys Gly LeuAsp Lys Leu Arg Phe Ser Val Val Gly Gln 385 390 395 400 Ser Lys Glu AlaAla Asn Glu Trp Ile Glu Lys Leu Lys Leu Ala Gly 405 410 415 Ala Asn PheTyr Ala Arg Val Val Lys Asp Ile Asp Cys Leu Ile Ala 420 425 430 Cys GlyGlu Leu Asp Asn Glu Asn Ala Glu Val Arg Lys Ala Arg Arg 435 440 445 LeuLys Ile Pro Ile Val Arg Glu Gly Tyr Ile Gly Glu Cys Val Lys 450 455 460Arg Thr Lys Cys Cys His Leu Ile Cys Ile Asn Trp Asn Ala Leu Glu 465 470475 480 Ser Ser Lys Gly Xaa Thr Val Thr Val Lys Val Lys Gly Arg Ser Ala485 490 495 Cys Ser Xaa Ser Pro Xaa Val Cys Lys Asn Thr Ala His Ile ProXaa 500 505 510 Xaa Trp Glu Lys His Ile Gln Cys Xaa Leu Lys His Val LeuThr Xaa 515 520 525 His Xaa Val Cys Thr Gly Tyr Tyr Val Leu Gln Ile IleGlu Gln Asp 530 535 540 Asp Gly Ser Glu Cys Tyr Val Phe Arg Lys Trp GlyArg Val Gly Ser 545 550 555 560 Glu Lys Ile Gly Gly Gln Lys Leu Glu GluMet Ser Lys Thr Glu Ala 565 570 575 Ile Lys Glu Phe Lys Arg Leu Phe LeuGlu Lys Thr Gly Asn Ser Trp 580 585 590 Glu Ala Trp Glu Cys Lys Thr AsnPhe Arg Lys Gln Pro Gly Arg Phe 595 600 605 Tyr Pro Leu Asp Val Asp TyrGly Val Lys Lys Ala Pro Lys Arg Lys 610 615 620 Asp Ile Ser Glu Met LysSer Ser Leu Ala Pro Gln Leu Leu Glu Leu 625 630 635 640 Met Lys Met LeuPhe Asn Val Glu Thr Tyr Arg Ala Ala Met Met Glu 645 650 655 Phe Glu XaaAsn Met Ser Glu Met Pro Leu Gly Lys Leu Ser Xaa Xaa 660 665 670 Asn IleGlu Xaa Gly Phe Glu Ala Leu Thr Xaa Xaa Xaa Xaa Leu Phe 675 680 685 GluGly His Arg Xaa Ser Ser Thr Gly Leu Xaa Glu Lys Ala Xaa Leu 690 695 700Leu Xaa Xaa Xaa Xaa Xaa Phe Ser Leu Leu Ser Leu Leu Phe Ile Leu 705 710715 720 Ile Leu Tyr Gly Met Arg Met Ile Ser Tyr Ser Lys Ala Lys Met Leu725 730 735 Glu Ala Leu Gln Asp Ile Glu Ile Ala Ser Lys Ile Val Gly PheAsp 740 745 750 Ser Asp Ser Asp Glu Ser Leu Asp Asp Lys Tyr Met Lys LeuHis Cys 755 760 765 Asp Ile Thr Pro Leu Ala His Asp Ser Glu Asp Tyr LysLeu Ile Glu 770 775 780 Gln Tyr Leu Leu Asn Thr His Ala Pro Thr His LysAsp Trp Ser Leu 785 790 795 800 Glu Leu Glu Glu Val Phe Ser Leu Asp ArgAsp Gly Glu Leu Asn Lys 805 810 815 Tyr Ser Arg Tyr Lys Asn Asn Leu HisAsn Lys Met Leu Leu Trp His 820 825 830 Gly Ser Arg Leu Thr Asn Phe ValGly Ile Leu Ser Gln Gly Leu Arg 835 840 845 Ile Ala Pro Pro Glu Ala ProVal Thr Gly Tyr Met Phe Gly Lys Gly 850 855 860 Leu Tyr Phe Ala Asp LeuVal Ser Lys Ser Ala Gln Tyr Cys Tyr Val 865 870 875 880 Asp Arg Asn AsnPro Val Gly Leu Met Leu Leu Ser Glu Val Ala Leu 885 890 895 Gly Asp MetTyr Glu Leu Lys Lys Ala Thr Ser Met Asp Lys Pro Pro 900 905 910 Arg GlyLys His Ser Thr Lys Gly Leu Gly Lys Thr Val Pro Leu Glu 915 920 925 SerGlu Phe Val Lys Trp Arg Asp Asp Val Val Val Pro Cys Gly Lys 930 935 940Pro Val Pro Ser Ser Ile Arg Ser Ser Glu Leu Met Tyr Asn Glu Tyr 945 950955 960 Ile Val Tyr Asn Thr Ser Gln Val Lys Met Gln Phe Leu Leu Lys Val965 970 975 Arg Phe His His Lys Arg 980 <210> SEQ ID NO 3 <211> LENGTH:474 <212> TYPE: DNA <213> ORGANISM: Zea mays <220> FEATURE: <221>NAME/KEY: CDS <222> LOCATION: (1)...(474) <400> SEQUENCE: 3 aac aag atgcta tta tgg cac ggt tca agg ttg acg aat ttt gtg gga 48 Asn Lys Met LeuLeu Trp His Gly Ser Arg Leu Thr Asn Phe Val Gly 1 5 10 15 att ctt agtcaa ggg cta aga att gca cct cct gag gca cct gtt act 96 Ile Leu Ser GlnGly Leu Arg Ile Ala Pro Pro Glu Ala Pro Val Thr 20 25 30 ggc tat atg ttcggc aaa ggc ctc tac ttt gca gat cta gta agc aag 144 Gly Tyr Met Phe GlyLys Gly Leu Tyr Phe Ala Asp Leu Val Ser Lys 35 40 45 agc gca caa tac tgttat gtg gat agg aat aat cct gta ggt ttg atg 192 Ser Ala Gln Tyr Cys TyrVal Asp Arg Asn Asn Pro Val Gly Leu Met 50 55 60 ctt ctt tct gag gtt gcttta gga gac atg tat gaa cta aag aaa gcc 240 Leu Leu Ser Glu Val Ala LeuGly Asp Met Tyr Glu Leu Lys Lys Ala 65 70 75 80 acg tcc atg gac aaa cctcca aga ggg aag cat tcg acc aag gga tta 288 Thr Ser Met Asp Lys Pro ProArg Gly Lys His Ser Thr Lys Gly Leu 85 90 95 ggc aaa acc gtg cca ctg gagtca gag ttt gtg aag tgg agg gat gat 336 Gly Lys Thr Val Pro Leu Glu SerGlu Phe Val Lys Trp Arg Asp Asp 100 105 110 gtc gta gtt ccc tgc ggc aagccg gtg cca tca tca att agg agc tct 384 Val Val Val Pro Cys Gly Lys ProVal Pro Ser Ser Ile Arg Ser Ser 115 120 125 gaa ctc atg tac aat gag tacatc gtc tac aac aca tcc cag gtg aag 432 Glu Leu Met Tyr Asn Glu Tyr IleVal Tyr Asn Thr Ser Gln Val Lys 130 135 140 atg cag ttc ttg ctg aag gtgcgt ttc cat cac aag agg tag 474 Met Gln Phe Leu Leu Lys Val Arg Phe HisHis Lys Arg * 145 150 155 <210> SEQ ID NO 4 <211> LENGTH: 157 <212>TYPE: PRT <213> ORGANISM: Zea mays <400> SEQUENCE: 4 Asn Lys Met Leu LeuTrp His Gly Ser Arg Leu Thr Asn Phe Val Gly 1 5 10 15 Ile Leu Ser GlnGly Leu Arg Ile Ala Pro Pro Glu Ala Pro Val Thr 20 25 30 Gly Tyr Met PheGly Lys Gly Leu Tyr Phe Ala Asp Leu Val Ser Lys 35 40 45 Ser Ala Gln TyrCys Tyr Val Asp Arg Asn Asn Pro Val Gly Leu Met 50 55 60 Leu Leu Ser GluVal Ala Leu Gly Asp Met Tyr Glu Leu Lys Lys Ala 65 70 75 80 Thr Ser MetAsp Lys Pro Pro Arg Gly Lys His Ser Thr Lys Gly Leu 85 90 95 Gly Lys ThrVal Pro Leu Glu Ser Glu Phe Val Lys Trp Arg Asp Asp 100 105 110 Val ValVal Pro Cys Gly Lys Pro Val Pro Ser Ser Ile Arg Ser Ser 115 120 125 GluLeu Met Tyr Asn Glu Tyr Ile Val Tyr Asn Thr Ser Gln Val Lys 130 135 140Met Gln Phe Leu Leu Lys Val Arg Phe His His Lys Arg 145 150 155 <210>SEQ ID NO 5 <211> LENGTH: 530 <212> TYPE: DNA <213> ORGANISM: Zea mays<400> SEQUENCE: 5 ctcgtgcaag tcatgccggt cccctatcgc caaggaccag ctccgtcttggcaagatggt 60 tcaggcgtca cagttcgacg gcttcatgcc gatgtggaac catgccaggtgcatcttcag 120 caagaagaac cagataaaat ccgttgacga tgttgaaggg atagatgcacttagatggga 180 tgatcaagag aagatacgaa actacgttgg gagtgcctca gctggtacaagttctacagc 240 tgctcctcct gagaaatgta caattgagat tgctccatct gcccgtacttcatgtagacg 300 atgcagtgaa aagattacaa aaggatcggt ccgtctttca gctaagcttgagagtgaagg 360 tcccaagggt ataccatggt atcatgccaa ctgtttcttt gaggtatccccgtctgcaac 420 tgttgagaag ttctcaggct gggatacttt gtccgatgag gataagagaaccatgctcga 480 tcttgttaaa aaagatgttg gcaacaatga acaaaataag ggttccaagc530

We claim:
 1. An isolated DNA molecule comprising a nucleotide sequenceselected from the group consisting of: a) a nucleotide sequence encodinga poly ADP-ribose polymerase having the amino acid sequence set forth inSEQ ID NO. 2; b) the nucleotide sequence set forth in SEQ ID NO. 1; andc) a nucleotide sequence that is antisense to the full-length sequenceset forth in SEQ ID NO.
 1. 2. A chimeric nucleic acid sequencecomprising a promoter capable of driving expression of a nucleic acid ina platlet cell operably linked to a nucleotide sequence of claim
 1. 3.The chimeric nucleic acid sequence of claim 2, wherein the nucleotidesequence encodes a poly ADP-ribose polymerase having the amino acidsequence set forth in SEQ ID NO.
 2. 4. The chimeric nucleic acidsequence of claim 3, wherein said nucleotide sequence is the nucleotidesequence set forth in SEQ ID NO.
 1. 5. A vector comprising the chimericnucleic acid sequence of claim
 4. 6. A plant cell transformed with thechimeric nucleic acid sequence of claim
 4. 7. A transformed plantcomprising the chimeric nucleic acid sequence of claim
 4. 8. Thechimeric nucleic acid sequence of claim 2, wherein the nucleotidesequence is antisense to the full-length sequence set forth in SEQ IDNO.1.
 9. A vector comprising the chimeric nucleic acid sequence of claim8.
 10. A plant cell transformed with the vector of claim
 9. 11. Atransformed plant comprising the chimeric nucleic acid sequence of claim8.
 12. A transformed plant having incorporated into its genome a DNAmolecule, said molecule comprising a promoter capable of drivingexpression of a nucleic acid sequence in a plant cell operably linked toa nucleotide sequence selected from the group consisting of: a) anucleotide sequence encoding a poly ADP-ribose polymerase having theamino acid sequence set forth in SEQ ID NO. 2; b) the nucleotidesequence set forth in SEQ ID NO. 1; and c) a nucleotide sequence that isantisense to the full-length sequence set forth in SEQ ID NO.
 1. 13. Thetransformed plant of claim 12, wherein the nucleotide sequence encodes apoly ADP-ribose polymerase having the amino acid sequence set forth inSEQ ID NO.
 2. 14. The transformed plant or claim 13, wherein said codingsequence is the nucleotide sequence set forth in SEQ ID NO.
 1. 15. Thetransformed plant of claim 12, wherein the nucleotide sequence isantisense to the full-length sequence set forth in SEQ ID NO.
 1. 16. Thetransformed plant of claim 12, wherein said plant is a dicot.
 17. Thetransformed plant of claim 12, wherein said plant is a monocot.
 18. Thetransformed plant of claim 17, wherein said plant is maize.
 19. Seed ofthe plant of any one of claims 16-18.
 20. A method for modulating themetabolic state of a plant cell, said method comprising transformingsaid plant with a DNA construct, said construct comprising a promoterthat drives expression in a plant cell operably linked with a nucleotidesequence selected from the group consisting of: a) a nucleotide sequenceencoding a poly ADP-ribose polymerase having the amino acid sequence setforth in SEQ ID NO. 2; b) the nucleotide sequence set forth in SEQ IDNO. 1; and c) a nucleotide sequence that is antisense to the full-lengthsequence set forth in SEQ ID NO.
 1. 21. The method of claim 20, whereinthe nucleotide sequence encodes a poly ADP-ribose polymerase having theamino acid sequence set forth in SEQ ID NO.
 2. 22. The method of claim21, wherein said coding sequence is the nucleotide sequence set forth inSEQ ID NO. 1.